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Biomedical subjects

C Holt

Publications and source records attributed to C Holt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Comparative motility of X and Y chromosome-bearing bovine sperm separated on the basis of DNA content by flow sorting.

A combination of flow cytometric sperm sorting of X and Y chromosome-bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33,342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 x 10(6) sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37 degrees C) and their motion video recorded for 2 min using a magnification of x 100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33,342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR.

Animals↗
PubMed1998

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

Alleles↗
PubMed1998

Prophylaxis against hepatitis B recurrence following liver transplantation using combination lamivudine and hepatitis B immune globulin.

Patients undergoing liver transplantation for hepatitis B-related liver disease are prone to recurrence. The mainstay of prophylaxis has been passive immunotherapy with hepatitis B immune globulin (HBIG). Antiviral therapy with lamivudine has proven effective in lowering hepatitis B virus (HBV) DNA and improving histology in patients with hepatitis B infection; its role in prophylaxis against hepatitis B recurrence following liver transplantation is under investigation. Viral breakthrough and resistance, however, are a significant problem with monotherapy with either HBIG or lamivudine. The efficacy of combination lamivudine/HBIG prophylaxis has not been reported. Fourteen patients underwent transplantation for decompensated liver disease owing to hepatitis B. Lamivudine (150 mg p.o./d) was begun before transplantation in 10 patients, including 4 who were HBV DNA-positive. In addition, 1 patient was HBV DNA-positive when transplanted. HBIG was given perioperatively and continued thereafter; treatment with lamivudine was maintained or initiated at the time of transplantation and continued indefinitely. The median follow-up was 387 days. Actuarial 1-year patient and graft survival was 93% (1 patient died of unrelated causes). At a median interval of 28 days following lamivudine treatment, all 5 HBV DNA-positive patients cleared HBV DNA from the serum; 1 went on to clear hepatitis B surface antigen (HBsAg), before transplantation, at day 148 of lamivudine treatment. By the highly sensitive polymerase chain reaction (PCR), at a median of 346 days (range, 130-525 days) following transplantation, all 13 surviving patients had no detectable serum HBV DNA. Lamivudine suppresses HBV replication in patients awaiting liver transplantation. At a median follow-up of 1.1 years, combination prophylaxis with lamivudine and HBIG prevented hepatitis B recurrence following liver transplantation.

Adult↗
PubMed1998

Resolution of recurrent hepatitis B in two liver transplant recipients treated with famciclovir.

Recurrent hepatitis B infection after orthotopic liver transplantation remains problematic despite prophylaxis with hepatitis B immune globulin (anti-HBs IgG). Recently, famciclovir (an oral nucleoside analog) has been shown to have potent antiviral activity against hepatitis B in vitro as well as in patients with chronic hepatitis B. We present two patients who developed recurrent hepatitis B after orthotopic liver transplantation and were treated with famciclovir, 500 mg t.i.d. Both patients subsequently responded with marked improvement in biochemical liver tests and histology, with subsequent loss of hepatitis B surface antigen. Famciclovir is a useful agent in the treatment of hepatitis B in the liver transplant recipient.

2-Aminopurine↗
PubMed1997

Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis.

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Animals↗
PubMed1997

Effect of sampling procedure and strain variation in Listeria monocytogenes on the discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variates analysis.

The ability to discriminate successfully among cultures of all species of the Listeria genus by infrared spectroscopy in combination with canonical variate analysis was confirmed. The robustness of the method was demonstrated by showing that the separation of L. monocytogenes and L. grayi was hardly affected by variations in broth medium, incubation temperature, incubation time and cell washing procedure. Discrimination among 24 strains of L. monocytogenes according to serotype allowed two groups to be recognised, one comprising serotypes 4 and 4b and the other containing serotypes 1, 1/2b and 1/2c. When strain variation was included in the species discrimination model, the classification of all the L. monocytogenes strains was virtually 100% correct.

Culture Media↗
PubMed1997

Use of OKT3 is associated with early and severe recurrence of hepatitis C after liver transplantation.

OBJECTIVE: To determine whether increased immunosuppression is a contributor to poor outcome in hepatitis C-infected orthotopic liver transplant (OLT) recipients, we retrospectively analyzed the consequences of using OKT3 in our program for steroid-resistant rejection (SRR). METHODS: We compared the histological recurrence of HCV in two contemporary cohorts of OLT recipients. Group 1 consisted of HCV-positive patients who received OKT3 for SRR (n = 19). Group 2 (n = 33) consisted of age-, gender-, and initial immunosuppression-matched HCV-positive controls who were treated with at least one steroid pulse for acute cellular rejection but who did not require treatment with OKT3. Liver biopsies were obtained per protocol within the first month and as necessary to evaluate abnormalities in serum liver chemistries. RESULTS: Mean and median follow-up were comparable for the two groups. Recurrence of HCV was diagnosed by histological verification and was documented in 16 of 19 (84.2%) group 1 patients versus 17 of 33 (51.5%) group 2 patients (p = 0.03). The interval to recurrence was significantly shorter in patients who received OKT3 (p = 0.028). Logistic regression identified OKT3 as a significant risk factor for the recurrence of HCV within the first year post-OLT (p = 0.0004). The histological severity score, based on the most recent liver biopsy at long-term follow-up or the explant biopsy if the patient required retransplantation, was significantly higher in group 1. Moreover, cirrhosis was demonstrable in a greater proportion of allografts in patients who had received OKT3 at some point (26.3% vs. 6%, p = 0.028). Long-term follow-up revealed a trend toward higher alanine aminotransferase levels (p = 0.05) and total bilirubin (p = 0.08) in group 1 patients. CONCLUSIONS: Our data suggest that allograft hepatitis in patients with preexisting HCV occurs earlier and with greater severity in patients treated with OKT3 for SRR, compared with age-, gender-, and initial immunosuppression-matched contemporary controls. Treatment of SRR with OKT3 may jeopardize long-term allograft function and survival in HCV-infected recipients by enhancing viral hepatitis recurrence. Clearly, the recognition of recurrent HCV and differentiation from acute cellular rejection remains a crucial issue in managing the OLT recipient with HCV.

Acute Disease↗
PubMed1996

Graft loss following liver transplantation in patients with chronic hepatitis C.

Liver disease due to hepatitis C (HCV) is an increasingly frequent indication for orthotopic liver transplantation (OLT). The aim of the current study was to analyze the causes of graft loss following OLT for chronic hepatitis C and the longterm outcome following retransplantation in a large university program. Between January 1990 and December 1995, 1183 patients underwent primary OLT at our center. In 304 patients, HCV was diagnosed by seropositivity and/or polymerase chain reaction. Fifty-six (18.4%) of these patients underwent retransplantation. The 36 patients retransplanted for primary non-function were excluded from further analysis. The other indications for regrafting (>30 days following primary transplant) included hepatic artery thrombosis (5), chronic rejection (4), severe HCV recurrence (5), and other etiologies (6). The cumulative survival rates for the 248 patients who received 1 OLT (group 1) were 84% after one year and 75% after three years. The corresponding rates for the 20 non-PNF patients who were retransplanted (group 2) were 60% and 43%, respectively (P<.0001). Moreover, logistic regression analysis confirmed that patients in group 2 were more than 4 times likely to die than patients in group 1 (P<.0034; risk ratio, 4.2; 95% confidence interval 1.61 to 11.37). Patients undergoing retransplantation had a high incidence of serious infectious complications leading to mortality. Two additional patients with severe recurrent HCV died awaiting liver retransplantation. Eight of the 304 total patients (2.6%) transplanted for chronic HCV developed graft failure secondary to HCV recurrence and 6 of the 8 were retransplanted; 3 of the 6 patients retransplanted are alive without evidence of histologic recurrence (mean follow-up less than 1 year). In summary, despite the high frequency of recurrent histologic evidence of HCV following primary OLT (70% at 3 years), graft loss attributable solely to HCV is an infrequent finding. Retransplantation per se is a risk factor for a fatal outcome, and the indication for reOLT does not appear to impact ultimate outcome. Serious infectious complications were the leading cause of mortality in patients retransplanted. Furthermore, given the indolent natural history of HCV, longer follow-up is necessary to determine the ultimate rate of graft loss due to HCV recurrence.

Biopsy↗
PubMed1996

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract.

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.

Animals↗
PubMed1996

Ability of a beta-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters.

The ability of casein in the form of colloidal-sized casein micelles to modulate the phase separation of calcium phosphate during milk secretion is adapted to produce nanometre-sized particles of calcium phosphate stabilized by a casein phosphopeptide (nanoclusters). The nanoclusters were prepared from an undersaturated solution of salts and the peptide by raising the pH homogeneously from about 5.5 to 6.7 with urea plus urease. Chemical analysis and IR spectroscopy showed that they comprise an amorphous dicalcium phosophate bound to the phosphopeptide. Multinuclear NMR spectroscopy of the cluster solutions showed that the small ions and free peptide in the solution were in a state of dynamic exchange with the nanoclusters. The peptide is linked to the calcium phosphate through its sequence of phosphorylated residues, but, in a proportion of adsorbed conformational states, the termini retain the conformational freedom of the unbound peptide. The ability of casein to form nanoclusters in milk suggests a more general mechanism for avoiding pathological calcification and regulating calcium flow in tissues and biological fluids exposed to or containing high concentrations of calcium.

Adsorption↗
PubMed1996

Failure of ganciclovir prophylaxis to prevent allograft reinfection following orthotopic liver transplantation for chronic hepatitis B infection.

The effect of ganciclovir prophylaxis on reinfection of hepatic allografts by hepatitis B virus (HBV) was studied in 26 patients undergoing orthotopic liver transplantation (OLT) for decompensated cirrhosis due to HBV. Patients were randomized to receive either ganciclovir (6 mg/kg/day intravenously for a total of 100 days) or acyclovir (10 mg/kg every 8 hours intravenously until discharged and then 800 mg orally every 6 hours) for a total of 100 days after OLT as part of a study of prophylaxis against cytomegalovirus infection. All patients received hepatitis B immunoglobulin (HBIG), 10,000 units intravenously, during the anhepatic phase, daily for the first 7 days, after OLT, and then every 4 weeks for 6 months, Seven of 12 (58%) patients in the ganciclovir group developed recurrent HBV, compared with 6/14 (46%) of the acyclovir group (nonsignificant). No significant difference was observed in time to recurrent HBV in the ganciclovir group (mean 13.2 months) compared to the acyclovir group (mean 11 months). Our results suggest that ganciclovir administered prophylactically for 100 days after OLT does not prevent or delay graft reinfection by HBV.

Acyclovir↗
PubMed1996

Major psychological disorders in acute and chronic TMD: an initial examination.

This study assessed psychological disorders in 50 patients with chronic temporomandibular disorders and 51 patients with acute TMD. The results revealed a significant psychological comorbidity in both groups of patients. Both groups had high rates of psychopathology that exceeded the base rates of the general population. Such findings are in keeping with an integrated biopsychosocial model of TMD, and have significant implications for treatment of these patients.

Acute Disease↗
PubMed1995

Thermal denaturation of beta-lactoglobulin: effect of protein concentration at pH 6.75 and 8.05.

Previous work on the thermal denaturation of beta-lactoglobulin at about neutral pH and concentrations generally above 50 mg/ml has shown that the temperature of the maximum in the thermogram increases only slightly with concentration. Likewise, there is little if any concentration dependence at acid pH over a wide concentration range. However, so far as we are aware, no work has been described on the thermal denaturation of beta-lactoglobulin in the physiological range of protein concentration and pH appropriate to milk. We report measurements at pH 6.75 and 8.05 in the concentration range 2-120 mg/ml and show that below about 50 mg/ml the position of the maximum becomes strongly dependent on concentration, passing through a minimum near 25 mg/ml and increasing towards the lowest concentrations where measurements were practicable. Moreover, the narrow, well defined and nearly symmetrical thermal transition observed at high protein concentrations contrasts with a broader and more asymmetric curve at lower concentrations. An explanation for the behaviour seen at the lower protein concentrations is suggested, based on the temperature- and concentration-dependent dissociation of the beta-lactoglobulin dimer and an associated conformational transition. The position of the maximum in the thermogram has a marked dependence on the rate of heating down to the lowest rate investigated of 10 degrees C per hour, showing the importance of slow kinetic effects in the denaturation of this protein.

Animals↗
PubMed1995

Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein.

A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Base Sequence↗